The present application claims the benefit of priority under 35 U.S.C. xc2xa7371 to Patent Convention Treaty (PCT) International Application Serial No: PCT/JP00/02996, filed May 10, 2000, and its priority document Japanese patent application serial number 129365/1999, filed May 11, 1999. These applications are explicitly incorporated herein by reference in their entirety and for all purposes.
1. Technical Field
The present invention relates to a method and a device for fluorescence measurement. More particularly, the present invention relates to a method and a device for fluorescence measurement which is favored for reading a biochip on which fluorescence-labeled samples such as DNAs or proteins are arranged in a plane.
2. Background Art
In the fields of molecular biology and biochemistry, hybridization reaction between a nucleic acid or a protein having a known sequence and a target molecule contained in a sample is employed for searching useful genes or for diagnosing diseases. For this purpose, a biochip is used which has a plurality of sample spots on its surface for processing a mass number of samples in a short time. Each sample spot on the biochip has a different probe immobilized thereon. This biochip is placed into a reaction vessel together with a sample DNA to hybridize probes bound to the sample spots on the biochip with the fluorescence-labeled sample DNA. Then, the biochip is irradiated with excitation light to measure the fluorescent intensity of each sample spot with a fluorescence measurement device. Accordingly, a binding level between each probe and the sample DNA is obtained, which is then converted into a desired piece of information.
FIG. 5 is a schematic view showing a conventional fluorescence measurement device used for reading a biochip. The fluorescence measurement device radiates excitation light to each sample spot on the biochip and incorporates fluorescence emitted from each sample spot via an optical fiber bundle according to a luminous point scanning system.
The surface of the biochip 100 made of a slide glass or the like, has fluorescence-labeled sample spots 101 such as DNAs and proteins in a lattice arrangement. For example, microscopic spots with diameters of 50 xcexcm are arranged placing a space of about 100 xcexcm therebetween in a direction represented by an arrow Y. A chip-transporting motor 103 transports the biochip 100 in the parallel direction represented by the arrow Y. A laser beam 107 generated by a laser 105 reflects off a rotary mirror 109 and is guided to the surface of the biochip as a luminous point. The rotary mirror 109 is rotated by a motor 111 in a direction represented by an arrow R. The laser beam 107 linearly scans the surface of the biochip 100 in a direction represented by an arrow X. A motor controller 119 controls the chip-transporting motor 103 and the motor 111 as described above to run the laser beam in X-direction while continuously transporting the biochip in Y-direction to radiate the whole sample surface of the biochip 100.
The fluorescence emitted from each sample spot on the biochip is guided to photomultiplier tubes (PMTs) 115a and 115b via optical fiber bundles 113a and 113b, respectively. The incident ends of the optical fiber bundles 113a and 113b are arranged in lines corresponding to the scanning line of the laser beam on the surface of the biochip 100. The other ends of the optical fiber bundles 113a and 113b extend to the PMTs 115a and 115b, respectively. Optical filters 117a and 117b are provided between the optical fiber bundles 113a and 113b and the PMTs 115a and 115b, respectively, so that only the fluorescent wavelength of interest is read by the PMTs 115a and 115b. The output from the PMT 115a and 115b is sent to a data processor 121 for data processing. In this manner, a plurality of light receiving systems with optical filters of different wavelength transmitting ranges are provided to allow polychromatic reading.
The light receiving systems of the above-described conventional fluorescence measurement device receive not only the fluorescence from the sample but also excitation laser beam reflected or scattered off the sample surface. Since the amount of the sample is microscopic, the quantity of excitation light incident to the light receiving system is so many times greater than the quantity of the fluorescence incident to the same. Thus, the optical filter needs to have a narrow wavelength transmitting range to eliminate the excitation light, in which case the fluorescence that needs to be detected is often cut-off as well. Since the number of the optical fiber bundles used for incorporating fluorescence needs to satisfy at least the number of the samples along the beam scanning direction, it is disadvantageous in terms of cost and it requires precise mechanism and adjustment for aligning optical axes.
Furthermore, since the light-receiving angle of the optical fibers is small and thus small amount of fluorescence is incorporated from the sample, an S/N ratio tends to be low. In order to increase the S/N ratio, there has been an attempt to receive only the fluorescence from the sample by using a pulsed laser and acoustooptic modulator (AOM) to attenuate the intensity of the excitation laser beam immediately after exciting the sample. However, pulsed lasers are expensive, and AOM merely attenuates the intensity of the laser beam by {fraction (1/1000)} and cannot completely prevent the excitation laser beam from contaminating.
The aim of the present invention is to solve the above-described problems concerned with the conventional technique and to provide a method and a device for fluorescence measurement with high detection accuracy with a simple structure by using a relatively inexpensive laser such as a continuous-wave laser (CW laser) as an excitation light source.
According to the present invention, an excitation light radiating section and a fluorescence detecting section are provided spatially separated from each other. The sample irradiated with excitation light at the excitation light radiating section is transported to the fluorescence detecting section so that the fluorescence can be detected without being interrupted by the excitation light. Accordingly, an S/N ratio is enhanced and thus the above-described aim can be accomplished.
A method for fluorescence measurement of the invention comprises the steps of: irradiating a sample with excitation light from an excitation light source; transporting the sample irradiated with excitation light to an optical axis of a fluorescence detector; and detecting fluorescence incident on the fluorescence detector. The sample comprises a biopolymer labeled with a fluorescent substance.
According to this method, the sample irradiated with excitation light is transported, within a time shorter than the duration time of the fluorescence emitted from the sample, to an optical axis of the fluorescence detector, the optical axis directing towards a position shifted from the excitation light irradiation position. Since the excitation light reflected or scattered off the sample at the excitation light irradiation position is not incident upon the fluorescence detector, only the fluorescence may be detected with high sensitivity.
A device for fluorescence measurement according to the present invention comprises: an excitation light source; an excitation light radiating means for irradiating a sample with excitation light from the excitation light source; a fluorescence detector for detecting fluorescence emitted from the sample irradiated with excitation light, the fluorescence detector having an optical axis that does not, above the sample, cross with an optical axis of the excitation light radiating means; and a sample transporting means for transporting the sample irradiated with the excitation light of the excitation light radiating means to the optical axis of the fluorescence detector.
In the device for fluorescence measurement, an optical axis of an excitation light radiating means and an optical axis of a fluorescence detector do not cross with each other. The fluorescence of the sample is not measured under the excitation light radiating means but after the sample is transported to the fluorescence detector within a time shorter than the duration time of the fluorescence. By spatially separating the fluorescence irradiation position from the fluorescence detecting position, the excitation light reflected or scattered off the sample at the excitation light irradiation position is not incident upon the fluorescence detector. As a result, only the fluorescence can be detected with high sensitivity.
Preferably, the fluorescence detector is provided with a photodetector such as PMT and a confocal optical system in which the photodetecting surface of the photodetector and the sample surface are conjugate. The sample transporting means may rotatively transport the sample. A plurality of sets of excitation light sources, excitation light radiating means and fluorescence detectors may be provided for detecting fluorescence of different wavelengths.
The fluorescent substance used with the present invention preferably has long fluorescence duration time and preferably is an Eu (europium) complex such as 4,4xe2x80x2-bis(1xe2x80x3,1xe2x80x3,1xe2x80x3,2xe2x80x3,2xe2x80x3,3xe2x80x3,2xe2x80x3-heptafluoro-4xe2x80x3,6xe2x80x3-hexanedion-6xe2x80x3-yl)chlorosulfo-o-terphenyl (BHHCT), rhodamine, FITC, Cy3 and Cy5. For example, BHHCT is a fluorescent substance which emits fluorescence with a wavelength of 615 nm as irradiated with a wavelength of 340 nm, and which has a fluorescent half-life of 100-200 xcexcsec which is very long compared to those of conventional fluorescent substances (which are several tens of nsec). By utilizing this property, a high level of fluorescence can be obtained upon irradiating the sample with excitation light and transporting the sample for fluorescence detection, thereby dramatically enhancing an S/N ratio.